Diagnostic assays for the photometric determination of analytes in fluids, including turbidimetric, nephelometric and colorimetric assays, are common and well-known. Due to their easy one-step procedure and their short turn-around times such assays are ideal candidates for the application in automated analyzers. Today, highly automated spectrophotometric analyzers are used in the clinical diagnostics to perform photometric assays in a time- and cost-efficient manner. The workflow on the analyzer is characterized by a simple procedure without any separation or washing step, typically involving the following protocol: a) the sample (serum or plasma) containing unknown amounts of analyte and analyte-specific assay reagents are dispensed into a reaction cuvette, b) in the cuvette the sample and the reagents are allowed to incubate for a certain time period at a prescribed temperature, c) the photometer measures the optical signal of the assay solution in the cuvette which correlates with the amount of analyte in the sample.
Broad test menus based on turbidimetric, nephelometric or colorimetric assays are offered for the clinical chemistry analyzers, e.g., COBAS C (Roche Diagnostics GmbH). The detection of these assays on the COBAS C instruments is based on a photometer with a tungsten halogen lamp as irradiation source, a grating for generating monochromatic light and photodiode array (12 diodes yielding 12 wavelengths between 340 and 800 nm) as detector.
Often critical samples are submitted for routine biochemical testing at clinical laboratories which show interferences with the applied assays, thus leading to altered and wrong results.
When working with laboratory tests that use optical methods, like colorimetric and turbidimetric approaches, substances in the sample matrix that are colored or scatter the light usually cause interferences. Examples for such interfering substances are hemoglobin (hemolysis), bilirubin (icterus) and lipids (lipemia), which absorb or scatter the light at wavelengths that are commonly used for spectrophotometric tests.
Hemolysis is an important interference factor that is usually attributable to in vitro damage from erythrocytes by different factors, such as prolonged storage of the blood before separating the serum or plasma, shear forces by rapidly forcing blood through small needles, excessive agitation when mixing or the physical act of centrifugation and separation of serum. In vivo hemolysis occurs less frequently, but it has the same effect on laboratory tests. The mechanism by which hemolysis interferes with the testing procedures is the color interference by the released hemoglobin, although also leakage of analytes from damaged erythrocytes and chemical interactions between red blood cell components and analytes are also possible reasons. As consequence, falsely higher or falsely lower analyte concentrations may be obtained in clinical tests due to the hemoglobin interference. The sample can also be contaminated by constituents of other blood cells like leukocytes and platelets. For example, cell decay can result in changes in blood of patients with leukemia; the decay of platelets during coagulation results in higher concentrations of intracellular platelet constituents in serum.
Hemolysis can be further caused by biochemical, immunological, physical and chemical mechanisms. During blood transfusion, complement-dependent hemolysis may be caused by antibodies reacting with the major blood group antigens. Physical hemolysis is caused by destruction of erythrocytes by hypotonicity, e.g., dilution of blood with hypotonic solution, as well as decreased (vacuum) or increased pressure. Mechanical hemolysis can occur during the flow of blood through medical devices, e.g., catheters, heart valves in-vivo, and by inadequate centrifugation as well as elevated temperature in-vitro. Contaminating substances can also cause in-vitro hemolysis. Finally, detergents and other contaminating substances can cause hemolysis. After the separation of blood cells, hemolysis is detected by the red color of serum or plasma. At extracellular hemoglobin concentrations exceeding 300 mg/L (18.8 mmol/L), hemolysis is detectable by the red color of serum or plasma. Samples with therapeutic hemoglobin derivatives are always intensely red colored. Some analytical systems measure the extent of hemolysis by comparing the absorption of samples at two wavelengths. The absorption spectrum of the hemoglobin derived oxygen carriers used as blood substitutes does not differ substantially from that of natural hemoglobin.
Bilirubin is a yellow pigment produced by enzymatic degradation of hemoglobin. Studies on bilirubin interference were mostly based on experiments in which free bilirubin or water-soluble di-taurobilirubin was added to serum. Under certain conditions the bilirubin molecules differ qualitatively and quantitatively in their effects of interference. Conjugated bilirubin appears in urine, when present at increased concentrations in blood. In patients with proteinuria, bilirubin bound to albumin can also appear in urine. After intra-cerebral bleedings unconjugated (free) bilirubin causes xanthochromia of the cerebrospinal fluid. At increased permeability of the blood-brain barrier bilirubin bound to albumin can appear in the CSF. Bilirubin has a high absorbance between 340 nm and 500 nm wavelengths. Therefore spectrophotometric tests using these wavelengths show limitations because of the constantly high background absorbance caused by bilirubin. The apparent increase or decrease of a result by bilirubin interference is assay- and analyte concentration-dependent.
Lipemic samples are samples of blood, serum, or plasma that have a cloudy or milky appearance due to increased lipid content. Lipemic samples cannot be avoided as increased concentration of lipids is often secondary to other disease states such as: diabetes mellitus, ethanol use, chronic renal failure and pancreatitis. The presence of lipemia can interfere with many clinical chemistry tests by different mechanisms, the most frequent mechanism being the scattering of light by the lipids, mainly chylomicrons and very low density lipoproteins, VLDL. As consequence the determined analyte concentrations can be altered, depending on the applied wavelengths and the lipid content.
In conclusion, the presence of hemoglobin, bilirubin and lipids and other interfering substances in a sample can cause a positive or a negative interference in the measurement result of photometric assays aimed at the quantitation of a specific analyte. Depending on the magnitude of the interference, the results may lead to wrong interpretation and inappropriate intervention.
To overcome the drawbacks of the interferences caused by hemolysis, icterus and lipemia several methods are known in the literature. Lipemic, icteric and hemolytic interferences can be reduced by pretreatment of the sample in a pre-analytical process to remove the interfering substance, e.g., by high speed centrifugation in case of lipemic samples. However, such countermeasures increase the workload and reduce the cost- and time-efficiency; such countermeasures are also prone to errors in the sample handling.
Another strategy is to use other clinical tests which are not sensitive to interferences. This may be challenging since alternative tests may need another instrument platform not available in the laboratory; also there might be no alternative test available on the market.
A correction of interferences caused by lipemia, hemolysis and icterus by using a blanking procedure is an alternative to overcome the limitations. This involves the measurement of the sample absorbance, once suitably diluted, prior to adding the assay reagents. The absorbance measured is subtracted from the final absorbance. A strategy to realize this blanking procedure is the utilization of 2 different reagents (blank and assay reagents) and 2 cuvettes. This approach improves the results, but it suffers from one drawback, reducing the throughput of tests by half. Another method involves the sequential adding of the reagents into the cuvette: a first reading is taken after a set time; afterwards assay reagents are added and incubated; finally a second reading is made. However, only poor improvement is usually achieved with this procedure. Furthermore, the established assay protocols may not be compatible with the new initial dilution step of the sample required for the first reading.
Bichromatic analysis allows also correcting the analytical results and is often applied in automated laboratory tests. A secondary (side) wavelength is used to measure the interfering substance. The analyte to be determined does not absorb at this second wavelength. This measurement is then subtracted from that of the analyte. This assumes that the absorbance of the interfering substance is the same at both wavelengths, which rarely is the case. Therefore, the bichromatic principle will only yield slight improvements in reducing interferences. Additionally, it is possible to treat the interference by chemically eliminating the interfering substance, e.g., bilirubin with bilirubin oxidase, or vitamin C with ascorbic oxidase.
Furthermore, multi-channel analyzers are fully automated, computer-controlled systems designed for the analysis of routine chemistry assays, immunoassays, and therapeutic drugs, e.g., COBAS 6000 (Roche Diagnostics GmbH) uses spectrophotometry to perform kinetic, end-point and non-linear reactions. To a certain extent, the system, similar to most modern analyzers, reduces spectral interference effects by application of two-reagent procedures and bichromatic spectrophotometry. The quality of the sample can be determined by different methods. A common method is to run a serum index test on the lab analyzer which quantifies the amount of bilirubin, haemoglobin and lipids present in the sample. The implementation of HIL indicies improved the accuracy and the quality of the test results.
However, there are still many patient samples showing interferences by hemolysis, bilirubin and lipids leading to erroneous results even by using HLI-indicies or correction methods. Analytical interference by hemolysis, bilirubin and lipids with laboratory assays is the most common concern in laboratory medicine. These altered and wrong results may lead to incorrect interpretation, wrong diagnosis, and potentially inappropriate intervention and unfavorable outcome for the patients. As consequence many samples have to be pretreated in a pre-analytical step to remove the interfering substance and then re-measured in cases where the concentration of hemoglobin, bilirubin, and lipids exceed a specific cut-off level. Pretreatments and re-measurements cause additional expenses and loss of time, both factors being critical for laboratories performing those assays.